Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation

Seedling explants of three tomato (Lycopersicon esculentum) and four bell pepper (Capsicum annuum) cultivars consisting of the radicle, the hypocotyl and one cotyledon were obtained after removing the primary and axillary meristems. After 14 days of incubation on solid Murashige and Skoog (MS) medium without growth regulators, explants of both species regenerated multiple shoots on the cut surface (2.9–5.3 shoots per explant for tomato and 1.2–2.2 for bell pepper cultivars). After excision, the shoots were rooted on solid MS medium and acclimated to the greenhouse. This method was highly efficient in tomato and, particularly, in bell pepper, where plant regeneration is especially difficult. We used these explants to transform tomato with Agrobacterium tumefaciens containing a 35S-GUS-intron binary vector. As shown by GUS expression, 47% of the tomato explants produced transformed meristems, which differentiated into plants that exhibited a low (3%) tetraploidy ratio. Southern blots and analysis of inheritance of the foreign genes indicated that T-DNA was stably integrated into the plant genome. The use of this technique opens new prospects for plant transformation in other dicotyledoneous plants in which genetic engineering has been limited, to date, due to the difficulties in developing an efficient in vitro regeneration system.     

Efficient transformation and regeneration of carnation cultivars usingAgrobacterium

We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.     

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

High-frequency regeneration and transformation ofRaphanus savus

We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s (MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing 0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin.     

Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

Induction of root and shoot formation from root meristems ofPetunia hybrida

Summary A procedure has been developed for the induction of root or shoot formation from root meristems of germinated seeds ofPetunia hybrida. Root formation was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine (BA) (0–0.5 mg/l) and naphtaleneacetic acid (NAA) (0.05–2.0 mg/l). Induction of predominantly shoot formation was obtained on MS medium containing the following combinations of hormones (in mg/l): 0.05–0.5 NAA and 0.25–2.0 BA. Complete plant formation was obtained after rooting of the shoots on MS medium supplemented with IAA (0–2.0 mg/l) or NAA (0-0.5 mg/l).     

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25±2°C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l^–1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

Effect of gelling agents and antibiotics on adventitious bud regeneration from In vitro leaves of pear

Summary The effect of the type of gelling agent and of several antibiotics on the adventitious bud regeneration from in vitro leaves was tested on eight pear genotypes. The use of gellan gum (Phytagel™) in the medium instead of agar had a very strong positive effect on the rate of adventitious bud regeneration for all pear genotypes tested in this study. This gelling agent induced faster cell divisions than agar, thus more callus was produced on wound sites and subsequently more buds regenerated. Incubation on gellan gum medium during the first 20 d of bud induction was sufficient to induce a stimulatory effect on regeneration and limited the production of hyperhydric buds. In the prospect of Agrobacterium transformation, the effect of several antibiotics was tested. Cefotaxime (200 mg/l) plus ticarcillin/clavulanic acid (100 mg/l) could be used in the culture medium without affecting the frequency of bud regeneration. The inhibition of bud regeneration was obtained with different kanamycin concentrations according to the gelling agent in the medium. On gellan gum medium, a concentration of 100 mg/l of kanamycin was suitable. These conditions can be recommended for experiments on Agrobacterium-mediated transformation of pear, where bacterial inoculation and presence of antibiotics generally reduce and delay bud regeneration.     

Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.

Summary An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog basal medium - LSD least significant difference - NOS nopaline synthase promoter - NPTII neomycin phosphotransferase (EC 2.7.1.95) - CaMV35S cauliflower mosaic virus promoter - GUS beta-glucuronidase (EC 3.2.1.31) - LB Luria Broth base - CTAB hexadecil trimethyl ammonium bromide - PCR polymerase chain reaction - X-gluc 5-bromo-4-chloro-3-indolyl-glucuronide     

Agrobacterium-mediated genetic transformation of oilseed Brassica campestris: Transformation frequency is strongly influenced by the mode of shoot regeneration

Summary Protocols were developed for efficient shoot regeneration from hypocotyl and cotyledon explants of oilseed Brassica campestris (brown sarson) cv. Pusa Kalyani. These were used for genetic transformation by an Agrobacterium based binary vector carrying neomycin phosphotransferase (npt) gene and -glucuronidase (gus)-intron gene for plant cell specific expression. Transformed plants were recovered from hypocotyl explants at a frequency of 7–13%. Addition of silver nitrate markedly enhanced shoot regeneration in hypocotyl explants under non-selection conditions and was found to be an absolute requirement under selection conditions. Cotyledon explants, inspite of being more regenerative, proved to be highly refractory to transformation. Only two chimeric transformed shoots were obtained from more than 10,000 cotyledons treated with Agrobacterium. In hypocotyl explants, shoot regeneration occurred from the vascular parenchyma both with and without the intervention of callus phase. Only the shoot buds differentiating from callus tissue were positive for GUS activity. In cotyledons, shoot buds originated only directly from the vascular parenchyma, generally at a distance of about 450–625 from the cut surface. Such shoots were negative for GUS activity.     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa)

An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation using cotyledon explants of oriental melon (Cucumis melo L. var. makuwa) has been developed. All six oriental melon cultivars evaluated in the study showed a >90?% shoot regeneration frequency and produced 1.8?C3.6 shoots per cotyledon explant when cultured on Murashige and Skoog (MS) medium supplemented with 1.0?mg?L^?1 benzyladenine and 0.01?mg?L^?1 indoleacetic acid. Kanamycin (Km) and geneticin (Gt) in the shoot induction medium (SIM) were compared both qualitatively and quantitatively for their efficiency as a selection agent for the selection and regeneration of transgenic plants after Agrobacterium-mediated transformation. Shoot formation was completely inhibited at 50?mg?L^?1 Km and 10?mg?L^?1 Gt. Relatively high concentrations of both Gt and Km (>100?mg?L^?1 Km and >25?mg?L^?1 Gt) were necessary because large numbers of non-transgenic shoots survived during the selection process. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed using ??-glucuronidase (GUS), PCR and Southern blot analysis. Shoot regeneration frequencies were 41.2?% at 100?mg?L^?1 Km and 15.2?% at 30?mg?L^?1 Gt 8?weeks after transformation, whereas the transformation frequencies based on the PCR were 2.9 and 7.1?%, respectively, 16?weeks after transformation. These results demonstrate that a large portion of the regenerated shoots on SIM supplemented with 100?mg?L^?1 Km consisted of non-transformed or escaped shoots, indicating that 30?mg?L^?1 Gt is the more suitable for the selection and regeneration of transgenic plants in oriental melon.     

Potential application of urea-derived herbicides as cytokinins in plant tissue culture

Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%) with a mean number of 5 shoots in alfalfa, inC. forskohlii, the frequency of regeneration was 90% with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.     

An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.Abbreviations MS Murashige and Skoog - BAP 6-Benzylaminopurine - TDZ Thidiazuron     

The growth regulators used for bud regeneration and shoot rooting affect the competence for flowering and seed set in regenerated plants of protein peas

Summary The production of whole plants from explants of protein pea (Pisum sativum L.) using an efficient, reliable and rapid strategy, while maintaining trueness to type, will be required before regeneration can be exploited for genetic transformation. Seeds of the pea genotypes Terese, Solara, Frisson and P64 (a hypernodulating mutant line of Frisson) were surface-sterilized and imbibed overnight, whereafter embryo axes were dissected and germinated on hormone-free medium for 7–10 d. Hypocotyl sections lacking pre-existing meristems were harvested and cultured on a range of media with various concentrations and combinations of growth regulators in order to induce either caulogenesis or somatic embryogenesis. Differences in responsiveness were apparent between genotypes, but regeneration via caulogenesis was consistently more reliable than via the induction of somatic embryos. Few explants underwent somatic embryo production and their conversion into plants has remained elusive so far, irrespective of the genotype studied. Conversely, large numbers of buds were produced within 10 d by organogenesis, and healthy, rootable shoots were obtained. A clear relationship was observed between the growth regulators employed for bud regeneration and shoot rooting phases and the subsequent competence of the regenerated plants for flowering, pod formation and viable seed production.